EP1596192A1 - Trennungsverfahren von Proteinen durch Kapillarelektroforese und Pufferzusammensetzung für Kapillarelektroforese - Google Patents

Trennungsverfahren von Proteinen durch Kapillarelektroforese und Pufferzusammensetzung für Kapillarelektroforese Download PDF

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EP1596192A1
EP1596192A1 EP05291001A EP05291001A EP1596192A1 EP 1596192 A1 EP1596192 A1 EP 1596192A1 EP 05291001 A EP05291001 A EP 05291001A EP 05291001 A EP05291001 A EP 05291001A EP 1596192 A1 EP1596192 A1 EP 1596192A1
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additive
buffer
process according
alkyl
capillary electrophoresis
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EP05291001A
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French (fr)
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EP1596192B1 (de
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Frédéric Robert
Denis Simonin
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Sebia SA
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Sebia SA
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Priority to PL05291001T priority Critical patent/PL1596192T3/pl
Priority to SI200531650T priority patent/SI1596192T1/sl
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Priority to CY20121101244T priority patent/CY1113490T1/el
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44747—Composition of gel or of carrier mixture

Definitions

  • the present invention relates to a process for the separation of proteins and peptides by capillary electrophoresis as well as buffer compositions comprising an additive useful for this separation, in the presence of lipoproteins in the sample.
  • the protein profiles obtained comprise five or six fractions which correspond to the protein constituents that are the albumin fraction, the ⁇ 1 - and ⁇ 2 -globulin fractions, the ⁇ -globulin fraction, or the ⁇ 1 - and ⁇ 2 -globulin fractions. , and the ⁇ -globulin fraction. Each of these fractions has one or more serum proteins.
  • Such separations can be made in electrophoresis capillary, in particular, using analysis buffers and techniques such as described in US Patent Re 36,011, EP 518,475, or EP 1,229,325 and EP 1 258 724.
  • protein constituent is meant here not only the protein constituents that are fractions ⁇ 1 -; ⁇ 2 -; ⁇ - or ⁇ 1 - and ⁇ 2 -; and ⁇ -globulin, but also the lipoprotein constituents that are mainly ⁇ -lipoproteins, ⁇ -tipoproteins and pre ⁇ -lipoproteins also called HDL, LDL and VLDL for "High Density Lipoprotein", “Low Density Lipoprotein” and “Very Low Density Lipoprotein””.
  • the profiles are sometimes imprecise in particular because of these lipoproteins, and mainly ⁇ -lipoprotein and pre ⁇ -lipoprotein, which appear in the zone of the profiles corresponding to ⁇ 1 - and ⁇ 2 -globulins and ⁇ 1 -globulin.
  • anionic surfactant as an additive to the analysis buffer, it was possible to obtain an improved separation, and in particular a purified profile in the zone of ⁇ 1 -, ⁇ 2 - and ⁇ 1 -globulins of the electrophoretic profile.
  • anionic surfactants capable of hydrophobic interactions with one or more lipoprotein constituents, and in particular the hydrophobic residues of lipoproteins. They can provide this or these lipoprotein constituents one or more negative charges. They can modify, and in particular reduce the electrophoretic mobility compared to that of other protein constituents.
  • the profiles obtained in capillary electrophoresis may have purer peaks, free of shoulder especially with respect to the fractions ⁇ 1 and ⁇ 2 , as appears in the examples. This is of great interest for the exploitation of hyperlipemic serum sample profiles in particular, and is also of interest in the analysis of normolipemic samples.
  • the invention relates to a method of capillary electrophoresis free solution at alkaline pH for the analysis of samples with protein constituents including one or more lipoprotein constituent (s), in which the sample is introduced into a capillary tube containing a analysis buffer, said analysis buffer further comprising at least one additive of the anionic surfactant type capable of hydrophobic interaction with one (or more) lipoprotein component (s).
  • This additive is capable to give one or more of these lipoprotein constituent (s) negative charges and to modify their electrophoretic mobility, compared to that of other protein constituents, non-lipidic.
  • This step is usually followed by the separation of Protein constituents by migration and detection of constituents.
  • the invention also relates to a method for electrophoretically separating the protein constituents of samples comprising at least albumin and the ⁇ 1 -, ⁇ 2 - and ⁇ 1 -globulin fractions, as well as lipoproteins, in a buffer of assay, wherein the assay buffer further comprises at least one anionic surfactant additive capable of hydrophobic interaction with lipoproteins.
  • the present invention also relates to a method of electrophoretic separation, by capillary electrophoresis at alkaline pH in free solution, of the protein constituents of a liquid sample comprising lipoproteins, in which method the sample comprising said constituents is passed into a capillary containing a an assay buffer further comprising at least one additive capable of interacting specifically with lipoproteins;
  • the additive may be an anionic surfactant having a hydrophobic portion, such as a C 10 to C 20 alkyl chain, and an anionic moiety providing a strong negative charge at a pH greater than 9.
  • the additive of the anionic surfactant type is used in low concentrations in the buffer, so that the interaction remains low on albumin and other non-lipidic protein constituents and particularly focused on lipoproteins, what is called here "Specific to lipoproteins". Indeed, for such an interaction specific with lipoproteins, it is preferable to use the additive of the type anionic surfactant at low concentrations. These concentrations depend, for each anionic surfactant, on its affinity for lipoproteins, on the one hand, and its affinity for albumin and other non-lipidic protein constituents, on the other hand. Concentration Optimal is thus different for each surfactant.
  • It can be from the order of less than 1 mM in the buffer, for example of the order of 0.001 at 0.2 mM, preferably greater than 0.01 and less than 0.1 mM, example between 0.01 mM and 0.09 mM, for example.
  • the Applicant has found, in particular for SDS, that a concentration of approximately 0.05 mM does not cause sufficient displacement of the lipoprotein constituents and that a concentration greater than 0.2 mM deforms the profiles (decrease in ⁇ fractions). 2 and ⁇ 1 to 0.25 mM), and even leads to 0.5 mM, to a total deformation of the profiles.
  • the compounds useful as capillary electrophoresis analysis buffer additive of the invention capable of specific hydrophobic interaction with lipoproteins may be anionic surfactants such as those used in MECC (Micellar Electrokinetic Capillary Chromatography), but at a concentration significantly lower than their critical micellar concentration.
  • these compounds are used in free-solution EC as indicated above, there is a contribution of negative charges on the lipoproteins by hydrophobic interaction between the hydrophobic residues of these lipoproteins and the hydrophobic part of these compounds. where a slow migration of these lipoproteins compared to that of other proteins.
  • the invention relates to electrolyte compositions for capillary electrophoresis comprising, in an acceptable support, at least one buffer and an additive of the anionic surfactant type as defined above, capable of migrating the lipoproteins outwards. areas where they usually migrate, especially outside the zones of fractions ⁇ 1 , ⁇ 2 and ⁇ 1 .
  • the use of additives according to the invention allows a very improved separation of the protein fractions ⁇ 1 , ⁇ 2 and ⁇ 1 by displacement of the lipoproteins outside the area specific to these fractions. It thus makes it possible to improve the accuracy and precision of the quantitative analysis of serum proteins, as compared with analyzes performed with the usual buffers.
  • the additives are particularly useful for the analysis of biological samples rich in ⁇ -lipoprotein and pre ⁇ -lipoprotein.
  • kits for analyzing the protein constituents in a biological sample, comprising at least one analysis buffer and an additive of the anionic surfactant type, capable of migrating the lipoproteins out of the cells. zones where they usually migrate, especially outside the zones of fractions ⁇ 1 , ⁇ 2 and ⁇ 1 and / or comprising a hydrophobic part, such as a C 10 to C 20 alkyl chain, and an anionic part bringing a strong negative charge to a pH greater than 9 and / or one or more solution (s) for washing the capillaries and / or dilution strips and / or one (or more) diluent (s) of the sample to be analyzed.
  • the buffer and the additive (s) and diluent (s) may be stored separately to be mixed extemporaneously, or stored as a mixture.
  • This kit may include indications for use in performing the analysis.
  • FIG. 1 represents an electropherogram of a serum analyzed by capillary electrophoresis using a buffer according to EP 1 229 325.
  • FIG. 2 represents an electropherogram of the same serum analyzed by capillary electrophoresis using the same buffer, however, further comprising an anionic surfactant additive the invention.
  • Figures 3A, 3B, 3C and 3D represent the electrophoregrams of the same normolipemic serum, made by EC using the same buffer added with 0; 0.05; 0.07 and 0.25 mM SDS respectively.
  • Figures 4A, 4B, 4C and 4D show the electrophoregrams of the same hyperlipemic serum, made in EC in using the same buffer added with 0; 0.05; 0.07 and 0.25 mM SDS respectively.
  • the conditions for performing a capillary electrophoresis EC are known to those skilled in the art. They may usually include a washing the capillaries with a washing solution, washing with the buffer analysis, any dilution (s) of the sample, the injection of sample, migration and detection. These steps can be performed by automata.
  • Conditions for producing a capillary electrophoresis are for example the appropriate conditions to use the Capillarys PLC (SEBIA).
  • the hydrophobic alkyl chain may be composed of at least one C 10 to C 24 alkyl chain, branched or unbranched, comprising at least one linear part of about 10 carbon atoms, especially 10 to 20 carbon atoms.
  • this hydrophobic part may include residues or functions which do not substantially modify its hydrophobic character.
  • the anionic pole may consist of one or more of chemical groups or functions from the following list: sulfonates, carboxylates, sulfates, and phosphates.
  • Including anionic surfactants can be cited as the C 10 -C 24 -alkyl-mono-, di- or tri-sulfates, C 10 -C 24 -alkyl-mono-, di- or tri-sulphonates, C 10 -C 24 -alkyl-mono-, di- or tri-carboxylates, C 10 -C 24 -alkyl-mono, -di or tri-phosphates and C 10 -C 24 alkylcarboxy-sulfonates, -sulfates and -phosphates, and in particular C 10 -C 24 alkylsulfates.
  • C 10 -C 24 monoalkylsulfates particularly C 10 -C 20 alkylsulphates, and among these C 10 -C 16 alkylsulfates, are preferred.
  • Dodecyl sulphate and more are particularly preferred. precisely sodium dodecyl sulphate (SDS).
  • the alkyl radicals are preferably linear.
  • the additives of the anionic surfactant type defined above can also be used in a mixture.
  • additives of the anionic surfactant type can advantageously be used in the presence of other additives known to interact with albumin, by improving the distance between ⁇ 1 -globulin and albumin as described in document EP 1 229,325.
  • C 6 to C 10 alkylsulphonates are preferred and among C 6 to C 10 alkylsulphonates, octanesulphonate is particularly preferred.
  • sample By sample according to the invention is meant the sample biological material to be analyzed, previously diluted with a dilution solution appropriate or analysis buffer, for example, or pure.
  • any biological fluid can be analyzed healthy or sick patients.
  • human body fluids can be normal serum or not, and also haemolyzed blood, plasma, urine or cerebrospinal fluid.
  • samples of animal origin can be analyzed.
  • the samples can also be synthetic proteins, and the process of the invention can then have production control targets by example.
  • the additives according to the invention are particularly useful for serum analyzes, and the separation of serum proteins, into human samples.
  • the serum proteins to be separated are albumin and ⁇ 1 - fractions; ⁇ 2 -; ⁇ (or ⁇ 1 - and ⁇ 2 -); and ⁇ -globulin, and ⁇ -lipoproteins, ⁇ -lipoproteins and pre ⁇ -lipoproteins, particularly ⁇ -lipoproteins and pre ⁇ -lipoproteins.
  • These denominations may include the protein constituents of all subtypes of these classes.
  • any buffer can be used known analysis, adapted to the desired separation, and useful in electrophoresis in general, and capillary electrophoresis in particular.
  • AT examples, mention may be made of borate, phosphate and carbonate buffers, buffers based on amino acid and so-called biological buffers.
  • SEBIA Capillarys B1B2 + buffer
  • Bis-TRIS (2-bis [2-hydroxyethyl] amino-2-hydroxymethyl-1,3-propanediol), ADA (N- [2-acetamido] -2-iminodiacetic acid), ACES (2- [2-acetamino] -2-aminoethanesulfonic acid), PIPES (1,4-piperazinediethanesulphonic acid), MOPSO (3- [N-morpholino] -2-hydroxypropanesulfonic acid), Bis-TRIS PROPANE (1,3-bis [tris (hydroxymethyl) methylaminopropane]), BES (N, N-bis [2-hydroxyethyl] -2-aminoethanesulphonic acid), MOPS (3- [N-morpholino] propanesulfonic acid), TES (2- [2-hydroxy-1,1-bis (hydroxymethyl) ethylamino] ethanesulfonic acid), HEPES
  • the pH of the buffer of analysis is between 8 and 12, preferably between 9 and 11, and more preferably, a value of about 10.
  • the analysis buffers according to the invention may furthermore comprise at least one pH-modifying compound.
  • a modifier of pH it is possible to use a compound chosen from lithium hydroxide, sodium hydroxide, potassium hydroxide, rubidium hydroxide, cesium hydroxide, francium hydroxide, monohydroxide, di-, tri- or tetraalkylammonium having from 1 to 8 carbon atoms in the alkyl part.
  • the analysis buffers are used in the usual conditions and concentrations, namely of the order of 10 to 500 mM, preferably 20 to 400 mM.
  • the additives according to the invention are used in concentrations defined above, low in relation to the concentrations described in EP 1 229 325 in the context of their interaction with albumin. In general, it is in the range of 0.001 to 0.2 mM, preferably 0.01 to 0.09 mM, and in the case of SDS, it is less than one that would cause too much interaction with albumin and other non-lipidic protein constituents, ie which would be disruptive to too important the profile.
  • micellar concentration value Critique intervenes for additives that are surfactants.
  • ocltanesulfonate When ocltanesulfonate is used, its concentration in the buffer is of the order of 1 to 10 mM, and preferably 2.5 to 5 mM.
  • the buffer may include one or more clean additives to increase the ionic strength.
  • buffer additive capable of increasing the force ionic level of the electrolyte
  • chlorides, sulphates or sulphonates of alkali metals and mixtures thereof.
  • the sulfate is used.
  • the sodium, lithium and potassium salts are selected. From additives mentioned above, sodium and / or lithium sulphate is preferred.
  • the buffer compositions of the invention are prepared from usual way for analysis buffer compositions, namely by addition of the constituents in liquid form, or solid to be diluted, to a acceptable support.
  • the support is water, distilled or demineralized.
  • capillaries From the point of view of the materials used for the capillaries, these are usual in capillary electrophoresis. Thus, we can use fused silica capillaries. Their internal diameter can range from 5 to 2,000 .mu.m. In a preferred manner, according to the invention, capillaries of internal diameter less than 200 microns, preferably less than 100 microns. We preferably uses untreated inner surface capillaries. The specialist will be able to adapt the nature of the capillary and its size to the needs of analysis.
  • Capillary electrophoresis of clinical specimens is performed on an EC apparatus equipped with a fused silica capillary of diameter internal 25 microns. The detection is carried out at 200 nm. The samples are placed in the autosampler of the device Capillarys (SEBIA) and the samples are injected automatically by injection hydrodynamic. The separation of the samples is carried out in less than 5 minutes by applying an electric field of about 400 V / cm. The capillary is washed before each analysis with 0.25 M sodium hydroxide and then analysis buffer.
  • the chemicals used are analytical grade.
  • the 150 mM borate buffer is prepared by dissolving 9.3 g boric acid (molar mass: 61.83 g / mol) in 1 l of demineralized water, and 5.1 g of sodium hydroxide (molar mass: 40.0 g / mol). The final concentration is of 150 mM and the pH of 10.0.
  • the human serum is diluted 1/5 in assay buffer.
  • a borate assay buffer is prepared as above.
  • Electrophoresis was performed according to the above method on a hyperlipemic human serum (Triglycerides: 5.73 g / l).
  • the electrophoregram obtained has, from left to right, six successive peaks attributed respectively to the albumin and ⁇ 1 , ⁇ 2 , ⁇ 1 , ⁇ 2 , and ⁇ -globulin fractions.
  • Fraction % albumin 52.3
  • Alpha 1 9.7
  • Alpha 2 9.6
  • Beta 1 6.4
  • Beta 2 7.8
  • Electrophoresis was performed as in Example 1.
  • the electrophoregram obtained has, from left to right, six successive peaks attributed respectively to the albumin and ⁇ 1 , ⁇ 2 , ⁇ 1 , ⁇ 2 , and ⁇ -globulin fractions.
  • the separation between the two fractions is significantly improved. Indeed, the spike marked by an arrow and supported on the alpha-1 peak (corresponding to pre ⁇ -lipoproteins) is removed from the profile.
  • Electrophoresis of a normolipemic human serum was carried out as in the previous examples in adding to the SDS buffer at a concentration of 0.00; 0.05; 0.07 and 0.25 mM.
  • the electrophoregrams obtained from left to right have six successive peaks attributed respectively to albumin and ⁇ 1 , ⁇ 2 , ⁇ 1 , ⁇ 2 and ⁇ globulin fractions.
  • the following table shows the results obtained.
  • FRACTION % SDS concentration (mM) 0.00 fig.3A 0.05 fig.3B 0.07 Fig.3c 0.25 fig.3D albumin 50.4 50.5 49.1 52.4 ⁇ 1 6.5 5.2 6.0 7.3 ⁇ 2 16.2 17.9 18.3 16.5 ⁇ 1 6.2 5.0 4.9 4.1 ⁇ 2 5.4 4.9 4.8 2.3 ⁇ 15.3 16.5 16.9 17.4
  • Slight lipid interference on albumin peak No interference Deformation fraction ⁇ 1 ; slight shoulder on albumin spike; decrease fraction ⁇ 2
  • Example 3 The procedure was as in Example 3 with a hyperlipemic human serum (triglycerides: 5.63 g / l).
  • the electrophoregrams obtained from left to right have six successive peaks attributed respectively to albumin and ⁇ 1 , ⁇ 2 , ⁇ 1 , ⁇ 2 and ⁇ globulin fractions.
  • the following table shows the results obtained.

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EP05291001A 2004-05-10 2005-05-10 Trennungsverfahren von Proteinen durch Kapillarelektroforese und Pufferzusammensetzung für Kapillarelektroforese Expired - Lifetime EP1596192B1 (de)

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Application Number Priority Date Filing Date Title
PL05291001T PL1596192T3 (pl) 2004-05-10 2005-05-10 Sposób rozdzielania białek za pomocą elektroforezy kapilarnej i kompozycje buforowe do elektroforezy kapilarnej
SI200531650T SI1596192T1 (sl) 2004-05-10 2005-05-10 Postopek za ločenje proteinov s kapilarno elektroforezo in pufrski sestavki za kapilarno elektroforezo
CY20121101244T CY1113490T1 (el) 2004-05-10 2012-12-20 Μεθοδος διαχωρισμου πρωτεϊνων με τριχοειδη ηλεκτροφορηση και συνθεσεις ρυθμιστικου για τριχοειδη ηλεκτροφορηση

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FR0405039 2004-05-10
FR0405039A FR2869995B1 (fr) 2004-05-10 2004-05-10 Procede ameliore de separation de proteines par electrophorese capillaire et compositions de tampon pour electrophorese capillaire

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EP1596192B1 EP1596192B1 (de) 2012-10-10

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US (1) US7906001B2 (de)
EP (1) EP1596192B1 (de)
JP (1) JP4693484B2 (de)
CN (1) CN1727887B (de)
AR (1) AR048735A1 (de)
BR (1) BRPI0501751B1 (de)
CY (1) CY1113490T1 (de)
DK (1) DK1596192T3 (de)
ES (1) ES2397156T3 (de)
FR (1) FR2869995B1 (de)
PL (1) PL1596192T3 (de)
PT (1) PT1596192E (de)
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US9121821B2 (en) * 2006-09-04 2015-09-01 National Institute Of Advanced Industrial Science And Technology Process for analyzing sample by capillary electrophoresis method
US20100155242A1 (en) * 2006-09-04 2010-06-24 Arkray, Inc. Method of Analyzing a Sample by Capillary Electrophoresis
JP4814945B2 (ja) * 2006-09-04 2011-11-16 独立行政法人産業技術総合研究所 キャピラリー電気泳動法による試料の分析方法
JP2009186445A (ja) * 2008-02-08 2009-08-20 Arkray Inc キャピラリー電気泳動法によるヘモグロビンの分析方法およびそれに用いる試薬
CN101339158B (zh) * 2008-08-28 2012-05-02 内蒙古蒙牛乳业（集团）股份有限公司 一种乳中β-酪蛋白含量的检测方法
CN101339159B (zh) * 2008-08-28 2012-05-02 内蒙古蒙牛乳业（集团）股份有限公司 一种乳中α-酪蛋白含量的检测方法
FR2947632B1 (fr) * 2009-07-01 2011-11-11 Sebia Sa Analyse et dosage d'hemoglobines glyquees par electrophorese capillaire, compositions tampon et kits pour electrophorese capillaire
WO2013188879A1 (en) * 2012-06-16 2013-12-19 Atherotech, Inc. Measurement of serum lipoproteins
EP3610851B1 (de) 2017-04-10 2022-02-16 Kao Corporation Hautreinigerzusammensetzung
EP3679364A1 (de) 2017-09-07 2020-07-15 Bristol-Myers Squibb Company Verfahren zur reinheitsbestimmung durch kapillarelektrophorese
CN109991303B (zh) * 2019-02-27 2023-10-03 北京工商大学 利用毛细管电泳技术快速鉴别单花蜂蜜的方法
JP2024064685A (ja) 2022-10-28 2024-05-14 アークレイ株式会社 試料分析方法、試料の製造方法、アルブミン及びγ－グロブリン含有試料希釈用希釈液、及び試料分析用キット
JP2024064686A (ja) 2022-10-28 2024-05-14 アークレイ株式会社 試料分析方法、キャピラリ電気泳動用溶液、及び試料分析用キット

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
US8702941B2 (en) 2009-12-25 2014-04-22 ARKARY, Inc. Method of analyzing hemoglobin by electrophoresis

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CN1727887B (zh) 2010-05-05
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FR2869995A1 (fr) 2005-11-11
JP4693484B2 (ja) 2011-06-01
US7906001B2 (en) 2011-03-15
ES2397156T3 (es) 2013-03-05
SI1596192T1 (sl) 2013-06-28
FR2869995B1 (fr) 2006-09-22
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CY1113490T1 (el) 2016-06-22
DK1596192T3 (da) 2013-01-07
AR048735A1 (es) 2006-05-17
PT1596192E (pt) 2013-01-23
CN1727887A (zh) 2006-02-01
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US20050274616A1 (en) 2005-12-15

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